Polar compounds such as pyridostigmine are used extensively in the treatment of patients with myasthenia gravis. Recently, pyridostigmine, in combined therapy with atropine and an oxime, was suggested as an antidote in organo phosphate poisonings as reported in Toxicol. Appl. Pharmacol., Vol. 43 (1978), page 207. In cases where patients received oral doses of pyridostigmine, considerable inter-subject variation of blood levels was reported in Neurology, Vol. 26, (1976) at page 536. Maximum plasma levels after similar oral doses administered to man range from 1 to 200 mg/ml. The quantitative isolation and accurate determination of the drug in biological fluids would result in establishing pharmacokinetic constants for pyridostigmine administered alone and in combination with other drugs.
Pyridostigmine is generally separated from plasma by extraction of an ion-pair complex with organic solvent. Quantitation has been reported by spectrophotometric in Z. Klin. Chem. Klin. Bio. Chem., Vol. 12 (1974), page 273, gas or liquid chromatographic analysis in Methods Find. Exp. Clin. Pharmacol., Vol. 2 (1980), page 77. Spectrophotometric methods are not sensitive enough to measure pyridostigmine in plasma after administration to man of 30 to 60 mg doses. Pohlmann and Cohan's method reported in J. Chromatogr., Vol. 131 (1977), page 297, based on electron-capture gas chromatography, measures picogram amounts, but lacks selectivity. In J. Chromatogr., Vol. 120 (1976), page 349, Chan et al. reported using an on-column gas chromatographic dequaternization method for selective and sensitive detection of nanogram amounts of pyridostigmine in plasma. Recently, two liquid chromatographic (LC) procedures have been introduced for determining pyridostigmine in J. Chromatogr., Vol. 164 (1979), page 399 and J. Chromatogr,, Vol. 183 (1980), page 193.